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Rudge, J.

Publications and source records attributed to Rudge, J..

2 recordsLinked to original sources
bioRxiv2025

Microparticle-Enabled Single Cell Multiparameter Electronic Immunophenotyping for Selective Electroporation

Electroporation (EP) is one of the leading non-viral intracellular delivery methods used in various applications across research and cell therapy development and manufacturing. Currently widely used bulk EP methods, while they offer scalability, cost efficiency and simplicity, cannot be used for targeted or selective delivery to a defined subset of a input cell population. Here, we present a Microparticle-Enabled Selectively Permeabilizing Impedance Cytometer (ME-SPICy), a microfluidic single-cell EP platform that enables targeted EP of selected cell subpopulations based on their surface markers. Antibody conjugated microparticles (MPs) are used to label selected cell subpopulations within a larger heterogenous sample. Using multifrequency impedance detection, ME-SPICy discriminates, in real-time, non-labeled and labeled cells within the mixed sample as they flow through a 3D printed biconical micro-aperture. This allows for the system to analyze if a cell is a target cell and selectively apply a low voltage (<16 V) for targeted single-cell EP. Simulations and experimental validation demonstrate that MP binding substantially alters cell impedance and phase signature, enabling accurate label-based discrimination. We demonstrated selective EP first using Jurkat cells by targeting either the labeled or non-labeled populations. Then we demonstrated targeted delivery to primary human lymphocytes within peripheral blood mononuclear cells. ME-SPICy achieved high precision, with 98% purity and >5 fold enrichment of lymphocytes in the electroporated cell population. This approach expands the capabilities of EP, offering a promising solution to decrease manufacturing complexity in both research and clinical cell engineering workflows

bioengineering↗
bioRxiv2025

Label-Free Targeted High Efficiency Electroporation with Single-Cell Feedback Control Using Focused Microscale Electric Fields

Efficient, safe, and cell-selective intracellular delivery remains a bottleneck for scalable and cost-effective manufacturing of cell therapies. Here, we introduce Selective Permeabilization using Impedance Cytometry (SPICy) that couples multifrequency single-cell impedance cytometry with real-time, feedback-controlled, low-voltage single-cell electroporation. Electric field focusing in a 3-D printed biconical micro-aperture confines both sensing and electroporation to a microscale zone, enabling continuous-flow operation and the use of low voltages (<15 V) for electroporation. Impedance spectra are captured for each single cell and machine-learning based analysis enabled accurately distinguishing cells in a label-free manner. Selectively triggered low-voltage electroporation achieved simultaneous high delivery efficiency (>80 %) and high (>90 %) cell viability. Delivery of a range of different cargo sizes (4-500 kDa), GFP mRNA expression, CRISPR-Cas9 based knock-out and delivery to a variety of different cell lines, primary human T cells and peripheral blood mononuclear cells (PBMCs) was also demonstrated. Using heterogenous or mixed samples, selective delivery to both cell lines, and primary immune cell subpopulations, from PBMCs, was demonstrated. SPICy thus provides a label-free, continuous flow, targeted non-viral platform for precision cell engineering.

bioengineering↗